One aspect of this project involves exploring the importance of the biosynthesis of various cofactors, cell wall assembly and turnover, and transcriptional and translational systems that maintain chromosomal integrity and replication in non-replicating (NR)-MTb. For example, we have previously demonstrated that NAD recycling and biosynthesis was critical for viability of MTb under both active replicating conditions as well as non-replicating persistence in vitro and in vivo by genetic and chemical genetic methods. We have now developed an enzyme assay for this enzyme that is suitable for high-throughput screening which we are using to screen small molecule libraries in an attempt to identify new leads for inhibitor design against this target. Analogs of initial hits from this screen have also been tested in this assay to further refine our understanding of the structure-activity relationships of potential inhibitors and their ability to inhibit growth of the organism and a knockdown mutant of the enzyme is currently underway to further explore the druggability of this target. We are continuing our systematic analysis of potential bottlenecks in the coenzyme A metabolic pathway to identify potential high-value drug targets. Conditional expression systems for each enzymatic step have been generated to identify those steps in the pathway that are most sensitive to inhibition. These chokepoints in coenzyme A biosynthesis are potentially the most sensitive to chemical inhibition and represent the best drug targets. The in vitro and in vivo validation of enzymes in this pathway through analysis of bacterial survival in response to target depletion has further corroborated the hypothesis that coenzyme A biosynthesis plays a role not only during replication but also during non-replicating persistence of the organism. Metabolomic studies of intracellular metabolite levels are further providing insight into the perturbations in the metabolic network that occur during down-regulation of the various steps in this pathway. Drug resistance in MTb is most commonly seen as a result of alterations in the binding site of a drug and its target. These mutations often have deleterious effects on the normal function of the mutated protein and organisms containing these mutant proteins often have significant growth impairment, an observation that is often used to argue that drug resistant TB presents only a relatively small threat. We have, however, unequivocally shown that compensatory mutations that ameliorate the cost of fitness associated with primary drug resistance mutations rapidly arise in vitro and in vivo. We have now demonstrated that heterogeneous drug treatment responses in patients can be ascribed to the evolution of distinct populations with different drug resistance in lung lesions where deep whole genome sequencing showed that each population acquired distinct mutations leading to differential drug resistance and fitness patterns. We hypothesized that the fitness cost associated with the primary drug resistance mechanism of each drug would have metabolic consequences in terms of the ability of the strain to become resistant to drugs targeting other essential processes in the organism. We are currently investigating the in vitro fitness of individual drug resistant strains with all clinically relevant drug resistance mechanisms and their ability to evolve resistance to second or third drug combinations. We are continuing our work on understanding metabolic consequences of inhibition of the folate pathway and how the spectrum of mutations elicited by folate pathway inhibitors affect in vitro versus in vivo fitness. Sequencing of clinical strains with resistance that had emerged in patients due to chemotherapy with PAS, showed that the majority of mutations were not in the target but in a folate consuming enzyme and that compensatory mutations emerged that likely generated strains with higher fitness in the human lung environment. In contrast, in vitro generated mutants map to the folylpolyglutamate synthase protein FolC conferring high level resistance with an apparent in vivo growth defect. The folC mutants are impaired in folate biosynthesis and are hyper-susceptible to other inhibitors of this pathway suggesting that synergistic drug combinations could prevent the emergence of resistance. Application of an affinity selectionmass spectrometry system for the discovery and characterization of proteinligand interactions, we have now identified novel inhibitors of the folate biosynthetic pathway that we are exploring for their potential to catastrophically impact folate dependent metabolism in the pathogen. Finally, we are continuing our work to understand how the metabolism of MTb is modulated in response to changes in the organisms microenvironment. We have demonstrated the dynamic changes in metabolite pools in response to hypoxic stress and how these changes reflect alterations in metabolic pathways. We are studying the uptake of metabolites by various transporters under these conditions and how uptake of metabolites competes with the susceptibility to specific novel growth inhibitors.